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Project Description

The 14-3-3 proteins are the prototype for a novel class of protein modules that can
recognize phosphoserine/threonine (pS/T)-containing motifs in a variety of signaling
proteins. To date, 14-3-3 proteins have been reported to bind more than 200 client proteins.
Through these interactions, 14-3-3 proteins play important roles in a wide range of
vital regulatory processes, such as Bad-induced apoptosis, Raf-1-mediated cell
proliferation, and Cdc25-regulated cell cycle progression. In addition to their
participation in diverse physiological processes, 14-3-3 proteins have been implicated in
a number of clinically important pathological conditions, such as neurodegenerative
disorders and cancers. Thus, such studies on the 14-3-3/client-protein interactions may
provide tremendous opportunities for therapeutic interventions. Therefore, chemical tools
would allow pharmacological probing of 14-3-3 function under various conditions.

Assay Description:

To develop small-molecule modulators of 14-3-3 proteins, a highly sensitive fluorescence
polarization (FP)-based 14-3-3 assay was designed and optimized. In this assay, the
interaction of 14-3-3 with a fluorescently labeled phosphopeptide from Raf-1 was
used as a model system. A simple 1-step “mix-and-measure” method was achieved
for analyzing 14-3-3 proteins. This is a solution-based, versatile method that can be
used to monitor the binding of 14-3-3 with a variety of client proteins. The 14-3-3 FP
assay is highly stable and has achieved a robust performance in a 384-well format
with a demonstrated signal-to-noise ratio of above 10 and a Z’ factor of above 0.7.
Because of its simplicity and high sensitivity, this assay is used for high-throughput
screening of 14-3-3 modulators.